Reversed-phase high-performance liquid chromatographic analysis of α-keto acid quinoxalinol derivatives. Optimization of technique and application to natural samples

A rapid, sensitive and selective method to quantify α-keto acids in natural samples is described. The basis of this method is the reaction of α-keto acids with o-phenylenediamine to form highly fluorescent quinoxalinol derivatives. These derivatives are separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and dtected fluorometrically. Information is provided concerning optimal derivatization and chromatographic conditions. Sample cleanup steps that are usually required prior to RP-HPLC analysis are eliminated. The method is reproducible (±1.90% at the 11 pmole level) and the fluorescent response is linearly related to α-keto acid concentration at both the μM and the nM level. Complete recoveries are obtained (≥98%) at the μM level. The detection limit is in the pmole to fmole range per injected acid. Applications to physiological and environmental samples are illustrated.